Technical Abstract:
Analysis of the polymethoxylated flavones (PMFs) in citrus products has been of interest for chemotaxonomic studies and because of their biological activity. They only occur in the oil glands which are located in the peel flavedo of intact fruit. PMFs from the peel are incorporated into citrus juices during the juice extraction process and normally occur at low ppm concentrations in commercial juice products. Due to the low concentrations of PMFs present in juice, sample preparation usually consists of liquid or solid phase extraction followed by concentration prior to analysis by either normal or reverse phase liquid chromatography. Solvent extracts of citrus peel normally do not need concentration due to the higher PMF concentrations present, but also contain many other components and solid phase extraction for sample clean-up facilitates PMF analysis. A sample preparation procedure utilizing a simple extraction protocol and analysis with in-line sample clean-up and trace enrichment using an automated switching valve has been developed to facilitate PMF analysis. The sample preparation method is similar to that developed for analysis of limonin in citrus products but utilizes a C-18 column for the analytical separation of the PMFs to optimize resolution. The method can be used for quantitative analysis of the PMFs in citrus juice, wet or dry peel, and citrus processing waste. Citrus juice preparation consists of simply diluting samples to contain 20% acetonitrile (v/v) while citrus solids are extracted by sonication in an appropriate amount of 20% acetonitrile. Prepared samples are then filtered and either 80-100mL juice or 5-20mL peel extract injected. Clean-up and PMF trace enrichment is performed with acetonitrile:water (20:80) to eliminate the flavanone glycosides and other polar components. The valve is then switched to elute the concentrated PMFs onto the C-18 analytical column with acetonitrile:water (40:60) and isocratic or gradient analysis depending on the desired application.